Lack of detectable endocytosis of B lymphocyte MHC class II antigens using an antibody-independent technique.
MHC class II complexes may intercept ingested foreign Ag as the nascent class II molecules exit the cell or as mature complexes recycle between the outer membrane and an internal compartment. To examine endocytosis of HLA-DR, we radiolabeled B lymphoblastoid cells, warmed the cells to allow internalization of membrane proteins, and then subjected viable cells to neuraminidase (NANAse)3 digestion. DR complexes were precipitated and analyzed by two-dimensional PAGE for shifts in isoelectric point signifying sensitivity to or protection from NANAse treatment. DR molecules were completely sensitive to the enzyme with or without specific antibody in the medium during the 37 degrees C incubation, suggesting that no detectable endocytosis had occurred. Control transferrin receptors, which readily internalize, showed marked protection. Assay sensitivity was measured by proportionate mixing of mock and NANAse-treated lysates; precipitated sialylated molecules were detectable at the 10% level. Monensin treatment to block recycling also failed to reveal any internally accumulated. NANAse-protected DR molecules. Measurements of turnover for surface-labeled DR complexes revealed a t 1/2 of approximately 36 h. We conclude that no large endocytically-derived pool of HLA-DR molecules exists within cells and suggest that the majority of mature DR molecules do not actively undergo internalization but reside at the cell surface after synthesis/transport for significantly long periods of time.
Davis
J E
JE
Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710.
Cresswell
P
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001402:B-Lymphocytes / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004705:Endocytosis; D006684:HLA-DR Antigens / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D009439:Neuraminidase / Q000378:metabolism; D013997:Time Factors
D000911:Antibodies, Monoclonal; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; C024138:I-E-antigen; D008985:Monensin; D009439:Neuraminidase